Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is one of the most frequent cancer predisposition syndromes and is characterized by early onset of colorectal cancers as well as cancers of the endometrium, stomach and upper urinary tract. Germline mutations in two of the five human mismatch repair (MMR) genes, hMSH2 and hMLH1, account for the vast majority of HNPCC cases, while mutations in the hPMS2 and hMSH6 MMR genes are rare. In addition, 5-15% of sporadic tumors of all types manifest the genetic instability (mutator phenotype) that is a hallmark of human mismatch repair defects. Since our initial discovery of hMSH2 and hMLH1, we have interrogated the biochemistry, molecular biology and function of the human mismatch repair proteins. In the last granting period we discovered two new MMR genes, extended the repertoire of protein associations which at least partially explained the distribution of mutations in HNPCC, assigned novel functions for the MMR proteins, and developed a new mechanism for MMR. In this continuing application we propose to: I.) fully characterize the functions and functional domains of hMSH2, hMSH3, HMSH6 and their heterodimeric forms hMSH2-hMSH3 and hMSH2-hMSH6, II.) fully characterize the functions and functional domains of hMLH1 and hPMS2 and their heterodimeric form hMLH1-hPMS2, III.) determine the functional alterations incurred by mutations found in HNPCC patients as well as specific mutational analysis of functional regions associated with the MMR proteins and their heterodimers, IV). analysis of the role(s) of hExoI in MMR and its association with hMSH2 and the role(s) of the major Thymine DNA Glycosylase (TDG) and its overlap with MMR components that perform the complete repair event. These events will detail the molecular and functional roles of the major human MMR protein components, analyze of the functional alterations with HNPCC, and should assist in the identification of molecular targets for therapeutic intervention and increased clinical efficacy in human cancer.